Methods and systems for in-vitro milk production

ABSTRACT

The disclosure relates to methods, systems and compositions for use in the production of milk. More specifically, the disclosure is directed to systems, compositions and methods for in-vitro production of milk using an array of mammary organoids seeded on tertiary-branched, resilient duct scaffolding.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/542,551 filed on Dec. 6, 2021, which is a continuation of U.S. patentapplication Ser. No. 17/383,495 filed on Jul. 23, 2021, now U.S. Pat.No. 11,236,299, which claims the benefit of priority under 35 USC §119(e) of U.S. Provisional Patent Application No. 63/075,476 filed onSep. 8, 2020. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

FIELD AND BACKGROUND OF THE INVENTION

The disclosure is directed to methods, systems and compositions for usein the production of milk More specifically, the disclosure is directedto systems, compositions and methods for in-vitro production of milkusing an array of mammary organoids seeded on tertiary-branched,resilient duct scaffolding.

The global dairy market, comprising the processing and harvesting ofanimal milk for human consumption, reached a value of US$718.9 Billionin 2019, and is typically sourced from cow, goat, buffalo, camel andsheep. With widespread demand for dairy products and their proactivefunction in the global food industry, dairy plays a crucial role in thegrowth of the economies worldwide.

Existing dairy milk alternatives, such as soy, almond, rice, or coconutmilk fall short both in flavor and in functionality; moreover, a largepart of the industrial and cultural significance of dairy milk stemsfrom its usefulness in derivative products, such as cheese, yogurt,cream, or butter. Non-dairy plant-based milks, while addressingenvironmental and health concerns (and while providing adequate flavorfor a small segment of the population), almost universally fail to formsuch derivative products when subjected to the same processes used fordairy milk.

Moreover, recent report from IATP noted, that as of 2017, the 13 topdairy companies' emissions grew 11% compared with 2015, corresponding toa 32.3 million metric ton increase in greenhouse gases equivalent to theemissions that would be released by adding an extra 6.9 million cars tothe road for a year.

Therefore, the need exists for methods, systems and compositions for usein the in-vitro production of milk, milk proteins (e.g., casein, whey)and milk products.

SUMMARY OF THE INVENTION

Disclosed, in various implementations, are systems, compositions andmethods for in-vitro production of milk using an array of mammaryorganoids. In other implementations, provided herein are methods forbio-printing of biostructures having a predetermined three dimensionalstructure with cells incorporated therein in a non-random two- and threedimensional pattern. In an exemplary implementation, provided herein isa system for in-vitro production of milk comprising: an array ofvessels, each vessel comprising a plurality of mammary organoids (MO); anutrient supply reservoir operable to feed each vessel; a milkcollection module, in communication with each vessel, operable tocollect milk produced by the MO; and a central processing module (CPM)in communication with the vessels' array, the nutrient supply reservoir,and the milk collection module, the CPM being in further communicationwith at least one processor and a memory storage device, storing thereona processor readable medium with a set of executable instructions,configured when executed to cause the at least on processor to controlthe operation of each of the vessels' array, the nutrient supplyreservoir, and the milk collection module, wherein the milk collecteddoes not comprise nutrients supplied.

In another exemplary implementation, provided herein is a method ofproducing milk in-vivo, implementable in a system comprising an array ofvessels, each vessel comprising a plurality of mammary organoids (MO); anutrient supply reservoir operable to feed each vessel; a milkcollection module, in communication with each vessel, operable tocollect milk produced by the MO; and a central processing module (CPM)in communication with the vessels' array, the nutrient supply reservoir,and the milk collection module, the CPM being in further communicationwith at least one processor and a memory storage device, storing thereona processor readable medium with a set of executable instructions,configured when executed to cause the at least on processor to controlthe operation of each of the vessels' array, the nutrient supplyreservoir, and the milk collection module, wherein the milk collecteddoes not comprise nutrients supplied, the method comprising: using thenutrient supply reservoir, contacting the MOs with the nutrients; andusing the milk collection module, collecting milk secreted by the MOs.

In yet another exemplary implementation, each vessel included in thesystems and the methods implemented thereby further comprise atertiary-branched, resilient scaffolding of hollow tubes in liquidcommunication with the nutrient supply reservoir and wherein theplurality of MOs are operably coupled to the resilient tertiary-branchedscaffolding.

In another exemplary implementation, the nutrient supply reservoirincluded in the systems and the methods implemented thereby furthercomprises: a first sub-reservoir with a composition comprising effectiveconcentration of estrogen and progesterone; a second sub-reservoir witha composition comprising lactation medium; a third sub-reservoir with acomposition comprising growth factor medium; and a fourth sub-reservoirwith a composition comprising contraction medium.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

For a better understanding of the systems, compositions and methods forin-vitro production of milk using an array of mammary organoids, withregard to the exemplary implementations thereof, reference is made tothe accompanying examples and figures, in which:

FIG. 1 is a schematic of an exemplary implementation of the system usedto implement the methods disclosed; and

FIG. 2 is a schematic of an exemplary implementation of thetertiary-branched resilient scaffolding tubes.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

Provided herein are implementations of systems, compositions and methodsfor in-vitro production of milk using an array of mammary organoids. Theexemplary implementations of the systems and compositions forimplementing the methods disclosed, are adapted to provide a continuousproduction of milk.

Mammary epithelial cells secrete milk constituents by several routes.Milk lipid is enveloped by a milk fat globule membrane (MFGM) derivedfrom the apical cell surface, and contains some of its constituentproteins. Soluble milk proteins are secreted by exocytosis. Threedimensional mammary epithelial cells culture (organoids) can be preparedfrom tissue of non-lactating, late-pregnant cows by, for examplecollagenase digestion and typical commercial isolation techniques (e.g.,centrifugation, HPLC and the like). Cells can be cultured directly onscaffolding, embedded in a reconstituted basement membrane and furthercultured in serum-free medium containing lactogenic hormones (e.g.,estrogen, progesterone and prolactin). The cells form multicellularstructures (mammary organoids) covered in matrix (“MOs”), and so long asthey are contacted with prolactin, nutrients and growth factors in acontrolled manner, do secrete milk for a period of between 10-21 days.

Accordingly and in an exemplary implementation, and as illustratedschematically in FIG. 1 , provided herein is system 10 for in-vitroproduction of milk comprising: an array 101 of vessels 102 i, eachi^(th) vessel comprising a plurality of mammary organoids (MO); nutrientsupply reservoir 103 operable to feed each i^(th) vessel; milkcollection module 104, in communication with each i^(th) vessel,operable to collect milk produced by the MO; and central processingmodule (CPM) 105 in communication with vessels' 102 i array 101,nutrient supply reservoir 103, and milk collection module 104, CPM 105being in further communication with at least one processor and a memorystorage device, storing thereon a processor readable medium with a setof executable instructions, configured when executed to cause the atleast on processor to control the operation of each of: vessels' 102 iarray 101, nutrient supply reservoir 103, and milk collection module104, wherein the milk collected does not comprise nutrients supplied.

In the context of the disclosure, the term mammary organoid meansthree-dimensional mammary epithelial cells inside collagen, or otherproteinaceous matrices, combining the advantages of easy manipulation of2D cellular systems with providing complex cell—cell and cell—ECMinteractions. The organoids are isolated in an exemplary implementationfrom bovine, goat, sheep, or other mammalian udder using typicalprocesses and coupled to the scaffolding such that each i^(th) vesselhas between about 250 and 2500 MOs per vessel.

In the systems and methods disclosed, each vessel is selectably (inother words, without affecting the function or structure of any othercomponent in the system) removable from the vessels' array. In otherwords, the vessel array is a modular bio-reactor where individualvessels (see e.g., 102 i, FIG. 1 ). The selectable removal is in anexemplary implementation beneficial to maintain optimal milk productionper vessel, allowing for replacement of vessels upon determination ofmalfunction, such as for example, contamination, cell division above agiven threshold (e.g., 15-24 divisions), MOs that have prematurelyundergone involution and the like. Furthermore, each vessel used in thesystems disclosed herein, comprises in certain implementations, atertiary-branched, resilient scaffolding of hollow tubes in liquidcommunication with the nutrient supply reservoir and wherein theplurality of MOs are operably coupled to the tertiary-branchedscaffolding (see e.g., FIG. 2 ).

In the context of the current disclosure, the term “scaffold”, or“scaffolding” refers in an exemplary implementation, to an engineeredplatform having a predetermined three dimensional structure, which mimicthe 3D environment of the mammary ductal system, provide short termmechanical support of the MOs, and provide an increased surface area forcells adhesion, proliferation, migration, and differentiation,eventually leading to accelerated tissue formation of the functionalMOs. Additionally or alternatively, “scaffolding” refers to a fabricatedsystems of conduits, sized adapted configured, to maintain fluidcommunication within the growing MOs to nutrients, buffer fluids,functionalizing fluids and other similar functional liquids. Asindicated, the scaffold can also be a composite scaffold. A “compositescaffold” refers to a scaffold platform which is engineered in order tosupport colonization and/or proliferation of two or more tissue typeswhich together comprise a “heterogeneous tissue”. In certainimplementations, the MOs are adhered to the resilient hollow tubes suchthat luminal cells are initially adhered to the tertiary-branchedresilient scaffold tubes and basally situated myoepithelial cells.

Likewise, the term “tertiary branched” refers to ducts that brunch frombranched ducts, all from a single trunk in each vessel, such that eachvessel is in liquid communication with a manifold feeding each vessel inthe array, connected to the nutrient reservoir. The duct diameter of themain trunk can be between about 0.5 mm and about 3 mm, and each primarybranch between about 0.5 mm and about 2 mm, a secondary branch can bebetween about about 0.5 mm and about 1.5 mm, and a tertiary branch canbe between about 0.5 mm and about 1 mm. In certain implementation, apump, included with the nutrient reservoir is sized and configured todeliver the nutrient composition to all branches, taking intoconsideration the pressure drop associated with the degree of branching.

In certain exemplary implementations, the scaffolding may be a resilientducts and can be, for example, poly(ethylenenaphthalate) (PEN),polyimide (e.g. KAPTONE® by DuPont), silicon ducts etc.

The milk collection module used in the systems disclosed can furtherinclude a vacuum source, such as a vacuum pump that can be a positivedisplacement pump, centrifufgal pump, or a Venturi tube. Likewise, thepump delivering the nutrients' composition to the vessels' array, can bea positive displacement pump, a diaphragm pump, or a triplex pump. Themilk collection module is in liquid communication with the vessels'array such that any milk secreted by the MOs, will not be in contactwith the nutrient stream, thus obviating the need to separate the milkfrom the nutrients.

In certain embodiments, each i^(th) vessel is embedded in gel, such as,for example, a composition comprising between about 50% (v/v) and about70% (v/v) laminin, between about 20% (v/v) and about 40% (v/v) collagen,and between about 5% (v/v) and 10% (v/v) of nidogen.

In an exemplary implementation, the nutrient supply reservoir used inthe systems disclosed, comprises: first sub-reservoir 1031 with acomposition comprising effective concentration of estrogen andprogesterone; second sub-reservoir 1032 with a composition comprisinglactation medium; third sub-reservoir 1033 with a composition comprisinggrowth factor medium; and fourth sub-reservoir 1034 with a compositioncomprising contraction medium (e.g., oxytocin).

Upon seeding of the scaffolding with the MO, estrogen and progesteroneare instrumental in inducing growth and morphogenesis of epithelium viainduction of paracrine signaling between mammary stroma and epitheliumcomprising the seeded MOs. Furthermore, lactation medium composition cancomprise prolactin (e.g., retrieved from the pituitary gland of the samemammal that was used to harvest the MOs, or a recombinant prolactin,configured to increase lactation over that associated with the prolactinisolated from the reference mammal), configured to acts directly onprolactin receptor on luminal cells included in the MOs and is operabletriggers alveoli maturation and lactogenic differentiation. Likewise,growth factor medium, can be for example basal organoid medium (BOM)supplemented with growth factors, such as, for example, FGF2, FGF7,FGF10, EGF, TGFα, WNT3A, R-spondin, or growth factor compositioncomprising one or more of the foregoing. Accordingly and in certainexemplary implementations, the set of executable instructions, isconfigured when executed to cause the at least on processor to, based ona predetermined sequence: controllably contact the vessel array with atleast one of: the composition of the first, second, third and fourth subreservoirs. In an exemplary implementation, the MOs are pretreated withFGF2, configured to accelerate lactogenic differentiation.

In addition, the vessels array, is enclosed in certain exemplaryimplementations, in a housing, operable to maintain the vessels in acontrolled atmosphere, meaning, an atmosphere having gaseous compositionthat is different than air, for example enriched in CO2, and reduced inNitrogen. Other sensors can be incorporated into the housing, forexample, thermometers, hygrometers and the like. Furthermore,determining optimal milk production and determination of the selectivereplacement of a particular vessel, can be done for example, bymonitoring the production of Csn2, and WaP proteins.

In the context of the disclosure, the term “operable” means the systemand/or the device (e.g., the nutrient dispensing pump) and/or theprogram, or a certain element, component or step is/are fully functionalsized, adapted and calibrated, comprising elements for, having theproper internal dimension to accommodate, and meets applicableoperability requirements to perform a recited function when activated,coupled or implemented, regardless of being powered or not, coupled,implemented, effected, actuated, realized or when an executable programis executed by at least one processor associated with the system,method, and/or the device.

In some exemplary implementations, the systems disclosed are used toimplement the methods provided. Accordingly and in yet another exemplaryimplementation, provided herein is a method of producing milk in-vivo,implementable in a system comprising an array of vessels, each vesselcomprising a plurality of mammary organoids (MO); a nutrient supplyreservoir operable to feed each vessel; a milk collection module, incommunication with each vessel, operable to collect milk produced by theMO; and a central processing module (CPM) in communication with thevessels' array, the nutrient supply reservoir, and the milk collectionmodule, the CPM being in further communication with at least oneprocessor and a memory storage device, storing thereon a processorreadable medium with a set of executable instructions, configured whenexecuted to cause the at least on processor to control the operation ofeach of the vessels' array, the nutrient supply reservoir, and the milkcollection module, wherein the milk collected does not comprisenutrients supplied, the method comprising: using the nutrient supplyreservoir, contacting the MOs with the nutrients; and using the milkcollection module, collecting milk secreted by the MOs, the methodfurther comprising controllably contacting the vessel array with atleast one of: the composition of the first, second, third and fourth subreservoirs. For example, the following differentiation, the system canbe configured to deliver a predetermined combination of growth factor(s)and other medium (e.g., lactation medium) together, to enhanceproduction of other milk components, such as fat globules, Csn2, and WaPproteins.

In relation to systems and methods disclosed, the term “operable” alsomeans the system and/or the circuit is fully functional and calibrated,comprises logic for, and meets applicable operability requirements toperform a recited function when executed by at least one processor

The term “comprising” and its derivatives, as used herein, are intendedto be open ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives.

All ranges disclosed herein are inclusive of the endpoints, and theendpoints are independently combinable with each other. “Combination” isinclusive of blends, mixtures, alloys, reaction products, and the like.The terms “a”, “an” and “the” herein do not denote a limitation ofquantity, and are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The suffix “(s)” as used herein is intended to include both thesingular and the plural of the term that it modifies, thereby includingone or more of that term (e.g., the image(s) includes one or moreimages). Reference throughout the specification to “one implementation”,“another implementation”, “an exemplary implementation,”, and so forth,when present, means that a particular element (e.g., feature, structure,and/or characteristic) described in connection with the implementationis included in at least one implementation described herein, and may ormay not be present in other implementations. In addition, it is to beunderstood that the described elements may be combined in any suitablemanner in the various implementations.

All ranges disclosed herein are inclusive of the endpoints, and theendpoints are independently combinable with each other. Furthermore, theterms “first,” “second,” and the like, herein do not denote any order,quantity, or importance, but rather are used to denote one element fromanother.

Likewise, the term “about” means that amounts, sizes, formulations,parameters, and other quantities and characteristics are not and neednot be exact, but may be approximate and/or larger or smaller, asdesired, reflecting tolerances, conversion factors, rounding off,measurement error and the like, and other factors known to those ofskill in the art. In general, an amount, size, formulation, parameter orother quantity or characteristic is “about” or “approximate” whether ornot expressly stated to be such. For example, “about” as used hereinmean a reasonable amount of deviation of the modified term such that theend result is not significantly changed. These terms of degree should beconstrued as including a deviation of at least ±5% or at least ±10% ofthe modified term if this deviation would not negate the meaning of theword it modifies.

Although the foregoing disclosure for systems, compositions and methodsfor in-vitro production of milk using an array of mammary organoidsseeded on tertiary-branched, resilient duct scaffolding has beendescribed in terms of some implementations, other implementations willbe apparent to those of ordinary skill in the art from the disclosureherein. Moreover, the described implementations have been presented byway of example only, and are not intended to limit the scope of theinventions. Indeed, the novel methods, programs, libraries and systemsdescribed herein may be embodied in a variety of other forms withoutdeparting from the spirit thereof. Accordingly, other combinations,omissions, substitutions and modifications will be apparent to theskilled artisan in view of the disclosure herein.

It is the intent of the Applicant(s) that all publications, patents andpatent applications referred to in this specification are to beincorporated in their entirety by reference into the specification, asif each individual publication, patent or patent application wasspecifically and individually noted when referenced that it is to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention. To the extent that section headings are used,they should not be construed as necessarily limiting. In addition, anypriority document(s) of this application is/are hereby incorporatedherein by reference in its/their entirety.

1-17. (canceled)
 18. A system for in-vitro production of milk componentscomprising: a. hollow tubes coated with mammary epithelial cells; b. anutrient supply reservoir operable to feed said mammary epithelialcells; c. a collection module for collecting said milk componentsproduced by said mammary epithelial cells, such that milk componentsproduced by said mammary epithelial cells are not in contact withnutrient stream from said nutrient supply reservoir.
 19. The system ofclaim 18, wherein said milk components collection module furthercomprises a vacuum source.
 20. The system of claim 18, wherein saidmammary epithelial cells are embedded in a gel.
 21. The system of claim20, wherein said gel is comprised of a composition comprising betweenabout 50% (v/v) and about 70% (v/v) laminin, between about 20% (v/v) andabout 40% (v/v) collagen, and between about 5% (v/v) and 10% (v/v) ofnidogen.
 22. The system of claim 18, wherein said mammary epithelialcells comprise post-parturition mammary epithelium cells.
 23. The systemof claim 18, wherein said nutrient supply reservoir comprises at leastone of: a first sub-reservoir with a composition comprising effectiveconcentration of estrogen and progesterone; a second sub-reservoir witha composition comprising lactation medium; a third sub-reservoir with acomposition comprising growth factor medium; and a fourth sub-reservoirwith a composition comprising contraction medium.
 24. The system ofclaim 18, further comprises a housing operable to maintain controlledatmosphere for said mammary epithelial cells.
 25. The system of claim18, wherein said mammary epithelial cells form multicellular structurescomprising luminal cells and myoepithelial cells and wherein saidluminal cells are adhered to the hollow tubes.
 26. The system of claim18, wherein said tubes are branched tubes.
 27. A method of producingmilk components, implementable in the system of claim 18, the methodcomprising: using said nutrient supply reservoir, contacting the mammarycells with nutrients; and using said milk components collection module,collecting said milk components secreted by said mammary cells.
 28. Themethod of claim 27, wherein each tube of said hollow tubes is selectablyremovable from said system.
 29. The method of claim 27, wherein saidmilk components collection module further comprises a vacuum source. 30.The method of claim 27, wherein said mammary epithelial cells areembedded in a gel.
 31. The method of claim 27, wherein said mammaryepithelial cells comprise post-parturition mammary epithelium cells. 32.The method of claim 27, wherein said nutrient supply reservoir comprisesat least one of: a first sub-reservoir with a composition comprisingeffective concentration of estrogen and progesterone; a secondsub-reservoir with a composition comprising lactation medium; a thirdsub-reservoir with a composition comprising growth factor medium; and afourth sub-reservoir with a composition comprising contraction medium.33. The method of claim 32, further comprises a housing operable tomaintain controlled atmosphere for said mammary epithelial cells. 34.The method of claim 27, wherein said mammary epithelial cells formmulticellular structures comprising luminal cells and myoepithelialcells and wherein said luminal cells are adhered to the hollow tubes.35. The method of claim 23, wherein said tubes are branched tubes.